Protein Chips for High Throughput Screening of Protein Activity

ABSTRACT

The present invention relates to protein chips useful for the large-scale study of protein function where the chip contains densely packed reaction wells. The invention also relates to methods of using protein chips to assay simultaneously the presence, amount, and/or function of proteins present in a protein sample or on one protein chip, or to assay the presence, relative specificity, and binding affinity of each probe in a mixture of probes for each of the proteins on the chip. The invention also relates to methods of using the protein chips for high density and small volume chemical reactions. Also, the invention relates to polymers useful as protein chip substrates and methods of making protein chips. The invention further relates to compounds useful for the derivatization of protein chip substrates.

This application is a continuation of U.S. patent application Ser. No.09/849,781 filed May 4, 2001, now U.S. Pat. No. 8,399,383, which claimsthe benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication No. 60/201,921, filed on May 4, 2000, and U.S. ProvisionalPatent Application No. 60/221,034, filed on Jul. 27, 2000, each of whichis incorporated herein by reference in its entirety.

This invention was made with government support under grant numbersDARPA/ONR R13164-41600099 and NIH (National Institutes of Health)RO1CA77808. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to protein chips useful for thelarge-scale study of protein function where the chip contains denselypacked reaction wells. The invention relates to methods of using proteinchips to assay simultaneously the presence, amount, and/or function ofproteins present in a protein sample or on one protein chip, or to assaythe presence, relative specificity, and binding affinity of each probein a mixture of probes for each of the proteins on the chip. Theinvention also relates to methods of using the protein chips for highdensity and small volume chemical reactions. Also, the invention relatesto polymers useful as protein chip substrates and methods of makingprotein chips. The invention further relates to compounds useful for thederivatization of protein chip substrates.

BACKGROUND OF THE INVENTION

The sequencing of entire genomes has resulted in the identification oflarge numbers of open reading frames (ORFs). Currently, significanteffort is devoted to understanding gene function by mRNA expressionpatterns and by gene disruption phenotypes. Important advances in thiseffort have been possible, in part, by the ability to analyze thousandsof gene sequences in a single experiment using gene chip technology.However, much information about gene function comes from the analysis ofthe biochemical activities of the encoded protein.

Currently, these types of analyses are performed by individualinvestigators studying a single protein at a time. This is a verytime-consuming process since it can take years to purify and identify aprotein based on its biochemical activity. The availability of an entiregenome sequence makes it possible to perform biochemical assays on everyprotein encoded by the genome.

To this end, it would be useful to analyze hundreds or thousands ofprotein samples using a single protein chip. Such approaches lendthemselves well to high throughput experiments in which large amounts ofdata can be generated and analyzed. Microtiter plates containing 96 or384 wells have been known in the field for many years. However, the size(at least 12.8 cm×8.6 cm) of these plates makes them unsuitable for thelarge-scale analysis of proteins because the density of wells is nothigh enough.

As noted above, other types of arrays have been devised for use in DNAsynthesis and hybridization reactions, e.g., as described in WO89/10977. However, these arrays are unsuitable for protein analysis indiscrete volumes because the arrays are constructed on flat surfaceswhich tend to become cross-contaminated between features.

Photolithographic techniques have been applied to making a variety ofarrays, from oligonucleotide arrays on flat surfaces (Pease et al.,1994, “Light-generated oligonucleotide arrays for rapid DNA sequenceanalysis,” PNAS 91:5022-5026) to arrays of channels (U.S. Pat. No.5,843,767) to arrays of wells connected by channels (Cohen et al., 1999,“A microchip-based enzyme assay for protein kinase A,” Anal Biochem.273:89-97). Furthermore, microfabrication and microlithographytechniques are well known in the semiconductor fabrication area. See,e.g., Moreau, Semiconductor Lithography: Principals Practices andMaterials, Plenum Press, 1988.

Recently devised methods for expressing large numbers of proteins withpotential utility for biochemical genomics in the budding yeastSaccharomyces cerevisiae have been developed. ORFs have been cloned intoan expression vector that uses the GAL promoter and fuses the protein toa polyhistidine (e.g., HISX6) label. This method has thus far been usedto prepare and confirm expression of about 2000 yeast protein fusions(Heyman et al., 1999, “Genome-scale cloning and expression of individualopen reading frames using topoisomerase I-mediated ligation,” GenomeRes. 9:383-392). Using a recombination strategy, about 85% of the yeastORFs have been cloned in frame with a GST coding region in a vector thatcontains the CUP1 promoter (inducible by copper), thus producing GSTfusion proteins (Martzen et al., 1999, “A biochemical genomics approachfor identifying genes by the activity of their products,” Science286:1153-1155). Martzen et al. used a pooling strategy to screen thecollection of fusion proteins for several biochemical activities (e.g.,phosphodiesterase and Appr-1-P-processing activities) and identified therelevant genes encoding these activities. However, strategies to analyzelarge numbers of individual protein samples have not been described.

Thus, the need exists for a protein chip in which the wells are denselypacked on the chip so as to gain cost and time advantage over the priorart chips and methods.

Citation or identification of any reference in Section II or any othersection of this application shall not be considered as admission thatsuch reference is available as prior art to the present invention.

SUMMARY OF THE INVENTION

The invention is directed to protein chips, i.e., positionallyaddressable arrays of proteins on a solid support, useful for thelarge-scale study of protein function wherein the protein chip containsdensely packed reaction wells. The invention is also directed to methodsof using protein chips to assay the presence, amount, and/orfunctionality of proteins present in at least one sample. The inventionalso is directed to methods of using the protein chips for high densityand small volume chemical reactions. Also, the invention is directed topolymers useful as protein chip substrates and methods of making proteinchips. The invention is directed to compounds useful for thederivatization of protein chips.

In one embodiment, the present invention provides a protein chipcomprising a flat surface, such as, but not limited to, glass slides.Dense protein arrays can be produced on, for example, glass slides, suchthat chemical reactions and assays can be conducted, thus allowinglarge-scale parallel analysis of the presence, amount, and/orfunctionality of proteins. In a specific embodiment, the flat surfacearray has proteins bound to its surface via a3-glycidooxypropyltrimethox-ysilane (GPTS) linker.

Furthermore, in another specific embodiment, the present inventionovercomes the disadvantages and limitations of the methods and apparatusknown in the art by providing protein chips with densely packed wells inwhich chemical reactions and assays can be conducted, thus allowinglarge-scale parallel analysis of the presence, amount, and/orfunctionality of proteins.

The general advantages of assaying arrays rather than one-by-one assaysinclude the ability to simultaneously identify many protein-probeinteractions, and to determine the relative affinity of theseinteractions. The advantages of applying complex mixtures of probes to achip include the ability to detect interactions in a milieu morerepresentative of that in a cell, and the ability to simultaneouslyevaluate many potential ligands.

In one embodiment, the invention is a positionally addressable arraycomprising a plurality of different substances, selected from the groupconsisting of proteins, molecules comprising functional domains of saidproteins, whole cells, and protein-containing cellular material, on asolid support, with each different substance being at a differentposition on the solid support, wherein the plurality of substancesconsists of at least 100 different substances per cm².

In another embodiment, the invention is a positionally addressable arraycomprising a plurality of different proteins, or molecules comprisingfunctional domains of said proteins, on a solid support, with eachdifferent protein or molecule being at a different position on the solidsupport, wherein the plurality of different proteins or moleculesconsists of at least 50% of all expressed proteins with the same type ofbiological activity in the genome of an organism.

In yet another embodiment, the invention is a positionally addressablearray comprising a plurality of different substances, selected from thegroup consisting of proteins, molecules comprising functional domains ofsaid proteins, whole cells, and protein-containing cellular material, ona solid support, with each different substance being at a differentposition on the solid support, wherein the solid support is selectedfrom the group consisting of ceramics, amorphous silicon carbide,castable oxides, polyimides, polymethylmethacrylates, polystyrenes andsilicone elastomers.

In still another embodiment, the invention is a positionally addressablearray comprising a plurality of different substances, selected from thegroup consisting of proteins, molecules comprising functional domains ofsaid proteins, whole cells, and protein-containing cellular material, ona solid support, with each different substance being at a differentposition on the solid support, wherein the plurality of differentsubstances are attached to the solid support via a3-glycidooxypropyltrimethoxysilane linker.

In another embodiment, the invention is an array comprising a pluralityof wells on the surface of a solid support wherein the density of thewells is at least 100 wells/cm².

The present invention also relates to a method of making a positionallyaddressable array comprising a plurality of wells on the surface of asolid support comprising the step of casting an array from amicrofabricated mold designed to produce a density of greater than 100wells/cm² on a solid surface. In another embodiment, the invention is amethod of making a positionally addressable array comprising a pluralityof wells on the surface of a solid support comprising the steps ofcasting a secondary mold from a microfabricated mold designed to producea density of wells on a solid surface of greater than 100 wells/cm² andcasting at least one array from the secondary mold.

In yet another embodiment, the invention is a method of using apositionally addressable array comprising a plurality of differentsubstances, selected from the group consisting of proteins, moleculescomprising functional domains of said proteins, whole cells, andprotein-containing cellular material, on a solid support, with eachdifferent substance being at a different position on the solid support,wherein the plurality of different substances consists of at least 100different substances per cm², comprising the steps of contacting a probewith the array, and detecting protein/probe interaction.

In still another embodiment, the invention is a method of using apositionally addressable array comprising a plurality of differentproteins, or molecules comprising functional domains of said proteins,on a solid support, with each different protein or molecule being at adifferent position on the solid support, wherein the plurality ofproteins and molecules consists of at least 50% of all expressedproteins with the same type of biological activity in the genome of anorganism, comprising the steps of contacting a probe with the array, anddetecting protein/probe interaction.

In another embodiment, the invention is a method of using a positionallyaddressable array comprising a plurality of different substances,selected from the group consisting of proteins, molecules comprisingfunctional domains of said proteins, whole cells, and protein-containingcellular material, on a solid support, with each different substancebeing at a different position on the solid support, wherein the solidsupport is selected from the group consisting of ceramics, amorphoussilicon carbide, castable oxides, polyimides, polymethylmethacrylates,polystyrenes and silicone elastomers, comprising the steps of contactinga probe with the array, and detecting protein/probe interaction.

In yet another embodiment, the invention is a method of using apositionally addressable array comprising a plurality of differentsubstances, selected from the group consisting of proteins, moleculescomprising functional domains of said proteins, whole cells, andprotein-containing cellular material, on a solid support, with eachdifferent substance being at a different position on the solid support,wherein the plurality of different substances are attached to the solidsupport via a 3-glycidooxypropyltrimethoxysilane linker, comprising thesteps of contacting a probe with the array, and detecting protein/probeinteraction.

In still another embodiment, the invention is a method of using apositionally addressable array comprising the steps of depositing aplurality of different substances, selected from the group consisting ofproteins, molecules comprising functional domains of said proteins,whole cells, and protein-containing cellular material, on a solidsupport, with each different substance being at a different position onthe solid support, wherein the plurality of different substancesconsists of at least 100 different substances per cm², contacting aprobe with the array, and detecting protein/probe interaction.

In a specific embodiment, the invention is a method of using apositionally addressable array comprising the steps of depositing aplurality of different substances, selected from the group consisting ofproteins, molecules comprising functional domains of said proteins,whole cells, and protein-containing cellular material, on a solidsupport, with each different substance being at a different position onthe solid support, wherein the plurality of different substancesconsists of at least 100 different substances per cm², and wherein thesolid support is a glass slide, contacting a probe with the array, anddetecting protein/probe interaction.

In another embodiment, the invention is a method of using a positionallyaddressable array comprising the steps of depositing a plurality ofdifferent proteins, or molecules comprising functional domains of saidproteins, on a solid support, with each different protein or moleculebeing at a different position on the solid support, wherein theplurality of different proteins or molecules consists of at least 50% ofall expressed proteins with the same type of biological activity in thegenome of an organism, contacting a probe with the array, and detectingprotein/probe interaction.

In another embodiment, the invention is a method of using a positionallyaddressable array comprising the steps of depositing a plurality ofdifferent proteins, or molecules comprising functional domains of saidproteins, on a solid support, with each different protein or moleculebeing at a different position on the solid support, wherein theplurality of different proteins or molecules consists of at least 50% ofall expressed proteins with the same type of biological activity in thegenome of an organism, and wherein the solid support is a glass slide,contacting a probe with the array, and detecting protein/probeinteraction.

In another embodiment, the invention is a method of making apositionally addressable array comprising the steps of casting an arrayfrom a microfabricated mold designed to produce a density of wells on asolid surface of greater than 100 wells/cm² and depositing in the wellsa plurality of different substances, selected from the group consistingof proteins, molecules comprising functional domains of said proteins,whole cells, and protein-containing cellular material, on a solidsupport, with each different substances being in a different well on thesolid support.

In another embodiment, the invention is a method of making apositionally addressable array comprising the steps of casting asecondary mold from a microfabricated mold designed to produce a densityof wells on a solid surface of greater than 100 wells/cm², casting atleast one array from the secondary mold, and depositing in the wells aplurality of different substances, selected from the group consisting ofproteins, molecules comprising functional domains of said proteins,whole cells, and protein-containing cellular material, not attached to asolid support, with each different substances being in a different well.

In yet another embodiment, the invention is a method of making apositionally addressable array comprising the steps of casting asecondary mold from a microfabricated mold designed to produce a densityof wells on a solid surface of greater than 100 wells/cm², casting atleast one array from the secondary mold, and depositing in the wells aplurality of different substances, selected from the group consisting ofproteins, molecules comprising functional domains of said proteins,whole cells, and protein-containing cellular material, with eachdifferent substance being in a different well.

A. DEFINITIONS

As used in this application, “protein” refers to a full-length protein,portion of a protein, or peptide. Proteins can be prepared fromrecombinant overexpression in an organism, preferably bacteria, yeast,insect cells or mammalian cells, or produced via fragmentation of largerproteins, or chemically synthesized.

As used in this application, “functional domain” is a domain of aprotein which is necessary and sufficient to give a desired functionalactivity. Examples of functional domains include, inter alia, domainswhich exhibit kinase, protease, phosphatase, glycosidase, acetylase,transferase, or other enzymatic activity. Other examples of functionaldomains include those domains which exhibit binding activity towardsDNA, RNA, protein, hormone, ligand or antigen.

As used in this application, “probe” refers to any chemical reagentwhich binds to a nucleic acid (e.g., DNA or RNA) or protein. Examples ofprobes include, inter alia, other proteins, peptides, oligonucleotides,polynucleotides, DNA, RNA, small molecule substrates and inhibitors,drug candidates, receptors, antigens, hormones, steroids, phospholipids,antibodies, cofactors, cytokines, glutathione, immunoglobulin domains,carbohydrates, maltose, nickel, dihydrotrypsin, and biotin.

Each protein or probe on a chip is preferably located at a known,predetermined position on the solid support such that the identity ofeach protein or probe can be determined from its position on the solidsupport. Further, the proteins and probes form a positionallyaddressable array on a solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a. Using the depicted recombination strategy, 119 yeast proteinkinases were cloned in a high copy URA3 expression vector (pEGKG) thatproduces GST fusion proteins under the control of thegalactose-inducible GAL10 promoter. GST::kinase constructs were rescuedinto E. coli, and sequences at the 5′-end of each construct weredetermined. The whole procedure was repeated when mutations werediscovered.

FIG. 1 b. Immunoblots of GST::kinase fusion proteins purified asdescribed. From three attempts, 106 kinase proteins were purified. Inspite of repeated attempts, the last 14 of 119 GST fusions wereundetectable by immunoblotting analysis, (e.g., Mps1 in the lane labeledwith star).

FIG. 2 a. The protein chips used in the kinase study were producedaccording to the following process, schematically depicted. Thepolydimethylsiloxane (PDMS) was poured over an acrylic master mold.After curing, the chip containing the wells was peeled away and mountedon a glass slide. Next, the surface of the chip was derivatized andproteins were then attached to the wells. Wells were first blocked with1% BSA, after which kinase, ³³P-γ-ATP, and buffer were added. Afterincubation for 30 minutes at 30° C., the protein chips were washedextensively, and exposed to both X-ray film and a Molecular DynamicsPhosphorImager, which has a resolution of 50 pin and is quantitative.For twelve substrates, each kinase assay was repeated at least twice;for the remaining five substrates, the assays were performed once.

FIG. 2 b. An enlarged picture of a protein chip.

FIG. 3. Protein chip and kinase assay results. Position 19 on every chipindicates the signal of negative control. Mps1 at position B4 showedstrong kinase activities in all 12 kinase reactions, although no visiblesignal could be detected on a western blot (FIG. 1 b).

FIG. 4 a-d. Quantitative analysis of protein kinase reactions. Kinaseactivities were determined using a Molecular Dynamics PhosphorImager,and the data were exported into an Excel spreadsheet. The kinase signalswere then transformed into fold increases by normalizing the dataagainst negative control. Signals of 119 kinases in four reactions areshown in log scale. The fold increases ranges from 1 to 1000 fold.

FIG. 4 e-h. To determine substrate specificity, specificity index (SI)was calculated using the following formula:SI_(ir)=F_(ir)/[(F_(i1)+F_(i2)+ . . . +F_(ir)/r], where i represents theidentity of the kinase used, r represents the identity of the substrate,and F_(ir) represents the fold increase of a kinase i on substrate rcompared with GST alone. Several examples of kinase specificity areshown when SI is greater than three.

FIG. 5 a. Phylogenetic tree derived from the kinase core domain multiplesequence alignment, illustrating the correlation between functionalspecificity and amino sequences of the poly(Tyr-Glu) kinases. Kinasesthat can use poly(Thr-Glu) as a substrate often map to specific regionson a sequence comparison dendrogram. The kinases that efficientlyphosphorylate poly(Tyr-Glu) are indicated by shading; two kinases thatweakly use this substrate are indicated in boxes. Rad53 and Ste7, whichcould not phosphorylate poly(Tyr-Glu), are indicated by asterisks. Asshown, 70% of these kinases lie in four sequence groups (circled).

FIG. 5 b. Structure of the rabbit muscle phosphorylase kinase (PHK)28.The positions of three basic residues and a methionine (Met) residue,which are preferentially found in kinases that can use poly(Tyr-Glu) asa substrate, are indicated. The asparagine (Asp) residue is usuallyfound in kinases that do not use poly(Tyr-Glu).

FIG. 6. Cross sectional views of lithographic steps in a process ofmaking protein chips.

a. A silicon wafer with two layers of silicon on either side of an oxidelayer.

b. The silicon wafer with a resistant mask layer on top.

c. The etching process removes silicon where the surface is unprotectedby the resistant mask. The depth of the etching is controlled by theposition of the oxide layer, i.e., the etching process does not removethe oxide layer.

d. The mask layer is removed, leaving the etched silicon wafer.

e. The protein chip material is applied to the mold.

f. After curing, the protein chip is removed from the mold. The proteinchip has an image that is the negative of the mold.

FIG. 7. Kinase/inhibitor assays on a protein chip. A human proteinkinase A (PKA), a human map kinase (MAPK), three yeast PKA homologs(TPK1, TPK2 and TPK3), and two other yeast protein kinases (HSL1 andRCK1) were tested against two substrates (i.e., a protein substrate forPKA and a commonly used kinase substrate, MBP) using differentconcentrations of a specific human PKA inhibitor, PKIα, or a MAPKinhibitor, SB202190. As shown in the figure, PKIα can specificallyinhibit PKA activities using both peptide and MBP as substrates.However, SB202190 did not show any inhibitory effect on PKA activity. Itis also interesting to note that PKIα did not inhibit the three yeastPKA homologs (TPK1, TPK2, TPK3) or the other two yeast protein kinasestested, HSL1 and RCK1.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to protein chips, i.e., positionallyaddressable arrays of proteins on a solid support, useful for thelarge-scale study of protein function, wherein the protein chip containsdensely packed reaction wells. A positionally addressable array providesa configuration such that each probe or protein of interest is locatedat a known, predetermined position on the solid support such that theidentity of each probe or protein can be determined from its position onthe array. The invention is also directed to methods of using proteinchips to assay the presence, amount, and/or functionality of proteinspresent in at least one sample. The invention also is directed tomethods of using the protein chips for high density and small volumechemical reactions. Also, the invention is directed to polymers usefulas protein chip substrates and methods of making protein chips. Theinvention further relates to compounds useful for the derivatization ofprotein chip substrate.

In one embodiment, the invention is a positionally addressable arraycomprising a plurality of different substances, selected from the groupconsisting of proteins, molecules comprising functional domains of saidproteins, whole cells, and protein-containing cellular material, on asolid support, with each different substance being at a differentposition on the solid support, wherein the plurality of differentsubstances consists of at least 100 different substances per cm². In oneembodiment, said plurality of different substances consists of between100 and 1000 different substances per cm². In another embodiment, saidplurality of different substances consists of between 1000 and 10,000different substances per cm². In another embodiment, said plurality ofdifferent substances consists of between 10,000 and 100,000 differentsubstances per cm². In yet another embodiment, said plurality ofdifferent substances consists of between 100,000 and 1,000,000 differentsubstances per cm². In yet another embodiment, said plurality ofdifferent substances consists of between 1,000,000 and 10,000,000different substances per cm². In yet another embodiment, said pluralityof different substances consists of between 10,000,000 and 25,000,000different substances per cm². In yet another embodiment, said pluralityof different substances consists of at least 25,000,000 differentsubstances per cm². In yet another embodiment, said plurality ofdifferent substances consists of at least 10,000,000,000 differentsubstances per cm². In yet another embodiment, said plurality ofdifferent substances consists of at least 10,000,000,000,000 differentsubstances per cm².

In another embodiment, the invention is a positionally addressable arraycomprising a plurality of different substances, selected from the groupconsisting of proteins, molecules comprising functional domains of saidproteins, whole cells, and protein-containing cellular material, on asolid support, with each different substance being at a differentposition on the solid support, wherein the plurality of differentsubstances consists of at least 100 different substances per cm², andwherein the solid support is a glass slide.

In another embodiment, the invention is a positionally addressable arraycomprising a plurality of different substances, selected from the groupconsisting of proteins, molecules comprising functional domains of saidproteins, whole cells, and protein-containing cellular material, on asolid support, with each different substance being at a differentposition on the solid support, wherein the plurality of differentsubstances consists of about 30 to 100 different substances per cm². Ina specific embodiment, said plurality of different substances consistsof 30 different substances per cm². In a particular embodiment, saidplurality of different substances consists of between 30 and 50different substances per cm². In another particular embodiment, saidplurality of different substances consists of between 50 and 100different substances per cm².

In various specific embodiments, the invention is a positionallyaddressable array comprising a plurality of different proteins, ormolecules comprising functional domains of said proteins, on a solidsupport, with each different protein or molecule being at a differentposition on the solid support, wherein the plurality of differentproteins or molecules consists of at least 50%, 75%, 90%, or 95% of allexpressed proteins with the same type of biological activity in thegenome of an organism. For example, such organism can be eukaryotic orprokaryotic, and is preferably a mammal, a human or non-human animal,primate, mouse, rat, cat, dog, horse, cow, chicken, fungus such asyeast, Drosophila, C. elegans, etc. Such type of biological activity ofinterest can be, but is not limited to, enzymatic activity (e.g., kinaseactivity, protease activity, phosphatase activity, glycosidase,acetylase activity, and other chemical group transferring enzymaticactivity), nucleic acid binding, hormone binding, etc.

A. Production of Protein Chips

The protein chips with densities of wells in an array of the presentinvention are preferably cast from master molds which have been stamped,milled, or etched using conventional microfabrication ormicrolithographic techniques. Preferably conventional microlithographictechniques and materials are utilized in the production of the mastermolds. Once a master mold has been produced, the master mold may then beused directly to mold the protein chips per se. Alternatively, secondaryor tertiary molds can be cast from the master mold and the protein chipscast from these secondary or tertiary molds.

The master mold can be made from any material that is suitable formicrofabrication or microlithography, with silicon, glass, quartz,polyimides, and polymethylmethacrylate (Lucite) being preferred. Formicrolithography, the preferred material is silicon wafers.

Once the appropriate master, secondary, or tertiary mold has beenproduced, the protein chip is cast. The protein chip can be cast in anysolid support that is suitable for casting, including either porous ornon-porous solid supports. Ceramics, amorphous silicon carbide, castableoxides that produce casts of SiO₂ when cured, polyimides,polymethylmethacrylates, and polystyrenes are preferred solid supports,with silicone elastomeric materials being most preferred. Of thesilicone elastomeric materials, polydimethylsiloxane (PDMS) is the mostpreferred solid support. An advantage of silicone elastomeric materialsis the ease with which they are removed from the mold due to theirflexible nature.

FIG. 6 illustrates an example of one method useful for realizinghigh-density arrays of wells on protein chips according to thisinvention. A silicon wafer with an oxide layer sandwiched between layersof silicon is provided (FIG. 6 a). Known as silicon-on-insulator or SOIwafers, these wafers are commonly available from wafer supply companies(e.g., Belle Mead Research, Belle Mead, N.J., and VirginiaSemiconductor, Fredericksburg, VA).

The silicon wafer is then patterned and etched via an etch process (FIG.6 b-d). The buried oxide layer acts as a very effective etch stop andresults in highly uniform etch depth across the wafer. Etch depth isindependent of the etch process and merely is determined by thethickness of the top silicon layer.

A wet chemical etch process (e.g., using KOH or tetra-methyl hydrazine(TMAH)) can be utilized. However, this technique is slightly moredependent on the crystal orientation of the silicon wafer. Thus, atechnique using a rarefied gas (typically SF₆) in a reactive ion etch(RIE) is preferred. RIE etching techniques are capable of realizinghighly anisotropic wells in silicon that are independent of the crystalorientation of the silicon wafer. The references G. Kovacs,Micromachined Transducers Sourcebook, Academic Press (1998) and M.Madou, Fundamentals of Microfabrication, CRC Press (1997) providebackground on etching techniques.

Both types of microlithography can be utilized on a single chip toobtain the desired combination of well shapes. Wet-chemical etching isan isotropic process which gives U-shaped wells, while RIE is ananisotropic process which gives square bottomed wells.

After etching the wafer to realize a master mold, it can be used to castprotein chips (FIG. 6 e-f). These structures can be the protein chips orthemselves be secondary or tertiary molds from which additional castingof protein chips occurs.

Thus, in one embodiment, a method of making a positionally addressablearray, comprising a plurality of wells on the surface of a solidsupport, comprises casting an array from a microfabricated mold designedto produce a density of wells on a solid surface of greater than 100wells/cm². In another embodiment, a method of making a positionallyaddressable array, comprising a plurality of wells on the surface of asolid support, comprises casting a secondary mold from saidmicrofabricated mold designed to produce a density of wells on a solidsurface of greater than 100 wells/cm² and casting at least one arrayfrom the secondary mold. In yet another embodiment, a method of making apositionally addressable array comprises covering the mold with a liquidcast material, and curing the cast material until the cast is solid. Theliquid cast material is preferably silicone elastomer, most preferablypolydimethylsiloxane. Into any of these positionally addressable arrays,a plurality of different substances, selected from the group consistingof proteins, molecules comprising functional domains of said proteins,whole cells, and protein-containing cellular material, can be depositedsuch that each different substance is found in a different well on thesolid support.

B. Features of Protein Chips

The protein chips of the present invention are not limited in theirphysical dimensions and may have any dimensions that are convenient. Forthe sake of compatibility with current laboratory apparatus, proteinchips the size of a standard microscope slide or smaller are preferred.Most preferred are protein chips sized such that two chips fit on amicroscope slide. Also preferred are protein chips sized to fit into thesample chamber of a mass spectrometer.

The wells in the protein chips of the present invention may have anyshape such as rectangular, square, or oval, with circular beingpreferred. The wells in the protein chips may have square or roundbottoms, V-shaped bottoms, or U-shaped bottoms. Square bottoms areslightly preferred because the preferred reactive ion etch (RIE)process, which is anisotropic, provides square-bottomed wells. The shapeof the well bottoms need not be uniform on a particular chip, but mayvary as required by the particular assay being carried out on the chip.

The wells in the protein chips of the present invention may have anywidth-to-depth ratio, with ratios of width-to-depth between about 10:1and about 1:10 being preferred. The wells in the protein chips of thepresent invention may have any volume, with wells having volumes ofbetween 1 μl and 5 μl preferred and wells having volumes of between 1 nland 1 μl being more preferred. The most preferred volume for a well isbetween 100 nl and 300 nl. For protein chips with very high densities ofwells, the preferred volume of a well is between 10 pl and 100 nl.

The protein chips of the invention can have a wide variety of density ofwells/cm². The preferred density of wells is between about 25 wells/cm²and about 10,000,000,000,000 wells/cm². Densities of wells on proteinchips cast from master molds of laser milled Lucite are generallybetween 1 well/cm² and 2,500 wells/cm². Appropriate milling toolsproduce wells as small as 100 μm in diameter and 100 μm apart. Proteinchips cast from master mold etched by wet-chemical microlithographictechniques have densities of wells generally between 50 wells/cm² and10,000,000,000 wells/cm², Wet-chemical etching can produce wells thatare 10 μm deep and 10 μm apart, which in turn produces wells that areless than 10 μm in diameter. Protein chips cast from master mold etchedby RIE microlithographic techniques have densities of wells generallybetween 100 wells/cm² and 25,000,000 wells/cm², RIE in combination withoptical lithography can produce wells that are 500 nm in diameter and500 nm apart. Use of electron beam lithography in combination with RIEcan produce wells 50 nm in diameter and 50 nm apart. Wells of this sizeand with equivalent spacing produces protein chips with densities ofwells 10,000,000,000,000 wells/cm². Preferably, RIE is used to producewells of 20 μm in diameter and 20 μm apart. Wells of this size that areequivalently spaced will result in densities of 25,000,000 wells/cm².

The microfabrication and microlithographic techniques described abovehave been used successfully to wet-chemically etch silicon wafers withwell sizes of 560 μm or 280 μm with spacing of about 1 mm. Thiscombination of wells and spacing produces arrays of about 410,000wells/cm² and about 610,000 wells/cm², respectively. When well size andspacing are equivalent, protein chips with about 3.19 million wells/cm²and 12.75 million wells/cm² are produced.

In one embodiment, the array comprises a plurality of wells on thesurface of a solid support wherein the density of wells is at least 100wells/cm². In another embodiment, said density of wells is between 100and 1000 wells/cm². In another embodiment, said density of wells isbetween 1000 and 10,000 wells/cm². In another embodiment, said densityof wells is between 10,000 and 100,000 wells/cm². In yet anotherembodiment, said density of wells is between 100,000 and 1,000,000wells/cm². In yet another embodiment, said density of wells is between1,000,000 and 10,000,000 wells/cm². In yet another embodiment, saiddensity of wells is between 10,000,000 and 25,000,000 wells/cm². In yetanother embodiment, said density of wells is at least 25,000,000wells/cm². In yet another embodiment, said density of wells is at least10,000,000,000 wells/cm². In yet another embodiment, said density ofwells is at least 10,000,000,000,000 wells/cm²,

C. Utilization of Protein Chips

In one embodiment, the present invention provides a protein chipcomprising a flat surface, such as, but not limited to, glass slides.Dense protein arrays can be produced on, for example, glass slides, suchthat chemical reactions and assays can be conducted, thus allowinglarge-scale parallel analysis of the presence, amount, and/orfunctionality of proteins (e.g., protein kinases). Proteins or probesare bound covalently or non-covalently to the flat surface of the solidsupport. The proteins or probes can be bound directly to the flatsurface of the solid support, or can be attached to the solid supportthrough a linker molecule or compound. The linker can be any molecule orcompound that derivatizes the surface of the solid support to facilitatethe attachment of proteins or probes to the surface of the solidsupport. The linker may covalently or non-covalently bind the proteinsor probes to the surface of the solid support. In addition, the linkercan be an inorganic or organic molecule. Preferred linkers are compoundswith free amines. Most preferred among linkers is3-glycidooxypropyltrimethoxysilane (GPTS).

In another embodiment, the protein chips of the present invention haveseveral advantages over flat surface arrays. Namely, the use of wellseliminates or reduces the likelihood of cross-contamination with respectto the contents of the wells. Another advantage over flat surfaces isincreased signal-to-noise ratios. Wells allow the use of larger volumesof reaction solution in a denser configuration, and therefore greatersignal is possible. Furthermore, wells decrease the rate of evaporationof the reaction solution from the chip as compared to flat surfacearrays, thus allowing longer reaction times.

Another advantage of wells over flat surfaces is that the use of wellspermit association studies using a fixed, limited amount of probe foreach well on the chip, whereas the use of flat surfaces usually involvesindiscriminate probe application across the whole substrate. When aprobe in a mixture of probes has a high affinity, but low specificity,the indiscriminate application of the probe mixture across the substratewill saturate many of the proteins with the high affinity probe. Thissaturation effectively limits the detection of other probes in themixture. By using wells, a limited amount of a probe can be applied toindividual wells on the chip. Thus, the amount of the probe applied toindividual proteins can be controlled, and the probe can be differentfor different proteins (situated in different wells).

Once a protein chip is produced as described above, it can be used toconduct assays and other chemical reactions. For assays, proteins orprobes will generally be placed in the wells. The presence or absence ofproteins or probes will be detected by the application of probes orproteins, respectively, to the protein chip. The protein-probeinteraction can be visualized using a variety of techniques known in theart, some of which are discussed below.

Proteins useful in this invention can be fusion proteins, in which adefined domain is attached to one of a variety of natural proteins, orcan be intact non-fusion proteins.

In another embodiment, protein-containing cellular material, such as butnot limited to vesicles, endosomes, subcellular organelles, and membranefragments, can be placed on the protein chip (e.g., in wells). Inanother embodiment, a whole cell is placed on the protein chip (e.g., inwells). In a further embodiment, the protein, protein-containingcellular material, or whole cell is attached to the solid support of theprotein chip.

The protein can be purified prior to placement on the protein chip orcan be purified during placement on the chip via the use of reagentsthat bind to particular proteins, which have been previously placed onthe protein chip. Partially purified protein-containing cellularmaterial or cells can be obtained by standard techniques (e.g., affinityor column chromatography) or by isolating centrifugation samples (e.g.,P1 or P2 fractions).

Furthermore, proteins, protein-containing cellular material, or cellscan be embedded in artificial or natural membranes prior to or at thetime of placement on the protein chip. In another embodiment, proteins,protein-containing cellular material, or cells can be embedded inextracellular matrix component(s) (e.g., collagen or basal lamina) priorto or at the time of placement on the protein chip. The proteins of theinvention can be in solution, or bound to the surface of the solidsupport (e.g., in a well, or on a flat surface), or bound to a substrate(e.g., bead) placed in a well of the solid support.

The placement of proteins or probes in the wells can be accomplished byusing any dispensing means, such as bubble jet or ink jet printer heads.A micropipette dispenser is preferred. The placement of proteins orprobes can either be conducted manually or the process can be automatedthrough the use of a computer connected to a machine.

Since the wells are self-contained, the proteins or probes need not beattached or bound to the surface of the solid support, but rather theproteins or probes can simply be placed in the wells, or bound to asubstrate (e.g., bead) that is placed in the wells. Other substratesinclude, but are not limited to, nitrocellulose particles, glass beads,plastic beads, magnetic particles, and latex particles. Alternatively,the proteins or probes are bound covalently or non-covalently to thesurface of the solid support in the wells. The proteins or probes can bebound directly to the surface of the solid support (in the well), or canbe attached to the solid support through a linker molecule or compound.The linker can be any molecule or compound that derivatizes the surfaceof the solid support to facilitate the attachment of proteins or probesto the surface of the solid support. The linker may covalently bind theproteins or probes to the surface of the solid support or the linker maybind via non-covalent interactions. In addition, the linker can be aninorganic or organic molecule. Preferred linkers are compounds with freeamines. Most preferred among linkers is3-glycidooxypropyltrimethoxysilane (GPTS).

Proteins or probes which are non-covalently bound to the well surfacemay utilize a variety of molecular interactions to accomplish attachmentto the well surface such as, for example, hydrogen bonding, van derWaals bonding, electrostatic, or metal-chelate coordinate bonding.Further, DNA-DNA, DNA-RNA and receptor-ligand interactions are types ofinteractions that utilize non-covalent binding. Examples ofreceptor-ligand interactions include interactions between antibodies andantigens, DNA-binding proteins and DNA, enzyme and substrate, avidin (orstreptavidin) and biotin (or biotinylated molecules), and interactionsbetween lipid-binding proteins and phospholipid membranes or vesicles.For example, proteins can be expressed with fusion protein domains thathave affinities for a substrate that is attached to the surface of thewell. Suitable substrates for fusion protein binding includetrypsin/anhydrotrypsin, glutathione, immunoglobulin domains, maltose,nickel, or biotin and its derivatives, which bind to bovine pancreatictrypsin inhibitor, glutathione-S-transferase, antigen, maltose bindingprotein, poly-histidine (e.g., HisX6 tag), and avidin/streptavidin,respectively.

D. Assays on Protein Chips

In one embodiment, the protein chips are used in assays by usingstandard enzymatic assays that produce chemiluminescence orfluorescence. Detection of various proteins and molecular modificationscan be accomplished using, for example, photoluminescence, fluorescenceusing non-protein substrates, enzymatic color development, massspectroscopic signature markers, and amplification (e.g., by PCR) ofoligonucleotide tags. Thus, protein/probe interaction can be detectedby, inter alia, chemiluminescence, fluorescence, radiolabeling, oratomic force microscopy. Probes binding to specific elements in thearray can also be identified by direct mass spectrometry. For example,probes released into solution by non-degradative methods, whichdissociate the probes from the array elements, can be identified by massspectrometry (see, e.g., WO 98/59361). In another example, peptides orother compounds released into solution by enzymatic digests of the arrayelements can be identified by mass spectrometry.

The types of assays fall into several general categories. As a firstexample, each well on the array is exposed to a single probe whosebinding is detected and quantified. The results of these assays arevisualized by methods including, but not limited to: 1) usingradioactively labeled ligand followed by autoradiography and/orphosphoimager analysis; 2) binding of hapten, which is then detected bya fluorescently labeled or enzymatically labeled antibody or highaffinity hapten ligand such as biotin or streptavidin; 3) massspectrometry; 4) atomic force microscopy; 5) fluorescent polarizationmethods; 6) rolling circle amplification-detection methods (Hatch etal., 1999, “Rolling circle amplification of DNA immobilized on solidsurfaces and its application to multiplex mutation detection”, Genet.Anal. 15 (2):35-40); 7) competitive PCR (Fini et al., 1999, “Developmentof a chemiluminescence competitive PCR for the detection andquantification of parvovirus B19 DNA using a microplate luminometer”,Clin Chem. 45 (9):1391-6; Kruse et al., 1999, “Detection andquantitative measurement of transforming growth factor-betal (TGF-betal)gene expression using a semi-nested competitive PCR assay”, Cytokine 11(2):179-85; Guenthner and Hart, 1998, “Quantitative, competitive PCRassay for HIV-1 using a microplate-based detection system”,Biotechniques 24 (5):810-6); 8) calorimetric procedures; and 9)biological assays, e.g., for virus titers.

As a second example, each well on the array is exposed to multipleprobes concurrently, including pooling of probes from several sources,whose binding is detected and quantified. The results of these assaysare visualized by methods including, but not limited to: 1) massspectrometry; 2) atomic force microscopy; 3) infrared red orfluorescently labeled compounds or proteins; 4) amplifiableoligonucleotides, peptides or molecular mass labels; and 5) bystimulation or inhibition of the protein's enzymatic activity.Information is gleaned from mixtures of probes because of thepositionally addressable nature of the arrays of the present invention,i.e., through the placement of defined proteins at known positions onthe protein chip, information about to what the bound probe binds isknown. If so desired, positions on the array that demonstrate bindingcan then be probed with individual probes to identify the specificinteraction of interest.

Useful information also can be obtained, for example, by incubating aprotein chip with cell extracts, wherein each well on the chip containsa reaction mix to assay an enzymatic activity of interest, and wherein aplurality of different enzymatic and/or substrate activities areassayed, and thereby identifying and measuring the cellular repertoireof particular enzymatic activities. Similarly, the protein chip can beincubated with whole cells or preparations of plasma membranes to assay,for example, for expression of membrane-associated proteins ormolecules, or binding properties of cell surface proteins or molecules.Cells, markers on a cell, or substances secreted by a cell that bind toparticular locations on the protein chip can be detected usingtechniques known in the art. For example, protein chips containingarrays of antigens can be screened with. B-cells or T-cells, wherein theantigens are selected from the group consisting of synthetic antigens,tissue-specific antigens, disease-specific antigens, antigens ofpathogens, and antigens of autologous tissues. The antigen or antigenicdeterminant recognized by the lymphocytes can be determined byestablishing at what position on the array activation of the cells byantigen occurs. Lymphocyte activation can be assayed by various meansincluding, but not limited to, detecting antibody synthesis, detectingor measuring incorporation of ³H-thymidine, probing of cell surfacemolecules with labeled antibodies to identify molecules induced orsuppressed by antigen recognition and activation (e.g., IgD, C3breceptor, IL-2 receptor, transferrin receptor, membrane class II MHCmolecules, CD23, CD38, PCA-1 molecules, HLA-DR), and identify expressedand/or secreted cytokines.

In another example, mitogens for a specific cell-type can be determinedby incubating the cells with protein chips containing arrays of putativemitogens, comprising the steps of contacting a positionally addressablearray with a population of cells; said array comprising a plurality ofdifferent substances, selected from the group consisting of proteins,molecules comprising functional domains of said proteins, whole cells,and protein-containing cellular material, on a solid support, with eachdifferent substance being at a different position on the solid support,wherein the density of different substances is at least 100 differentsubstances per cm²; and detecting positions on the solid support wheremitogenic activity is induced in a cell. Cell division can be assayedby, for example, detecting of measuring incorporation of ³H-thymidine bya cell. Cells can be of the same cell type (i.e., a homogeneouspopulation) or can be of different cell types.

In yet another example, cellular uptake and/or processing of proteins onthe protein chips can be assayed by, for example, using radioactivelylabeled protein substrates and measuring either a decrease inradioactive substrate concentration or uptake of radioactive substrateby the cells. These assays can be used for either diagnostic ortherapeutic purposes. One of ordinary skill in the art can appreciatemany appropriate assays for detecting various types of cellularinteractions.

Thus, use of several classes of probes (e.g., known mixtures of probes,cellular extracts, subcellular organelles, cell membrane preparations,whole cells, etc.) can provide for large-scale or exhaustive analysis ofcellular activities. In particular, one or several screens can form thebasis of identifying a “footprint” of the cell type or physiologicalstate of a cell, tissue, organ or system. For example, different celltypes (either morphological or functional) can be differentiated by thepattern of cellular activities or expression determined by the proteinchip. This approach also can be used to determine, for example,different stages of the cell cycle, disease states, altered physiologicstates (e.g., hypoxia), physiological state before or after treatment(e.g., drug treatment), metabolic state, stage of differentiation ordevelopment, response to environmental stimuli (e.g., light, heat),cell-cell interactions, cell-specific gene and/or protein expression,and disease-specific gene and/or protein expression.

Enzymatic reactions can be performed and enzymatic activity measuredusing the protein chips of the present invention. In a specificembodiment, compounds that modulate the enzymatic activity of a proteinor proteins on a chip can be identified. For example, changes in thelevel of enzymatic activity are detected and quantified by incubation ofa compound or mixture of compounds with an enzymatic reaction mixture inwells of the protein chip, wherein a signal is produced (e.g., fromsubstrate that becomes fluorescent upon enzymatic activity). Differencesbetween the presence and absence of the compound are noted. Furthermore,the differences in effects of compounds on enzymatic activities ofdifferent proteins are readily detected by comparing their relativeeffect on samples within the protein chips and between chips.

The variety of strategies of using the high density protein chips of thepresent invention, detailed above, can be used to determine variousphysical and functional characteristics of proteins. For example, theprotein chips can be used to assess the presence and amount of proteinpresent by probing with an antibody. In one embodiment, apolydimethylsiloxane (PDMS) chip of GST fusion proteins can be probed todetermine the presence of a protein and/or its level of activity. Theprotein can be detected using standard detection assays such asluminescence, chemiluminescence, fluorescence or chemifluorescence. Forexample, a primary antibody to the protein of interest is recognized bya fluorescently labeled secondary antibody, which is then measured withan instrument (e.g., a Molecular Dynamics scanner) that excites thefluorescent product with a light source and detects the subsequentfluorescence. For greater sensitivity, a primary antibody to the proteinof interest is recognized by a secondary antibody that is conjugated toan enzyme such as alkaline phosphatase or horseradish peroxidase. In thepresence of a luminescent substrate (for chemiluminescence) or afluorogenic substrate (for chemifluorescence), enzymatic cleavage yieldsa highly luminescent or fluorescent product which can be detected andquantified by using, for example, a Molecular Dynamics scanner.Alternatively, the signal of a fluorescently labeled secondary antibodycan be amplified using an alkaline phosphatase-conjugated or horseradishperoxidase-conjugated tertiary antibody.

Identifying substrates of protein kinases, phosphatases, proteases,glycosidases, acetylases, or other group transferring enzymes can alsobe conducted on the protein chips of the present invention. For example,a wide variety of different probes are attached to the protein chip andassayed for their ability to act as a substrate for particularenzyme(s), e.g., assayed for their ability to be phosphorylated byprotein kinases. Detection methods for kinase activity, include, but arenot limited to, the use of radioactive labels, such as ³³P-ATP and³⁵S-γ-ATP, or fluorescent antibody probes that bind to phosphoaminoacids. For example, whereas incorporation into a protein ofradioactively labeled phosphorus indicates kinase activity in one assay,another assay can measure the release of radioactively labeledphosphorus into the media, which indicates phosphatase activity. Inanother example, protease activity can be detected by identifying, usingstandard assays (e.g., mass spectrometry, fluorescently labeledantibodies to peptide fragments, or loss of fluorescence signal from afluorescently tagged substrate), peptide fragments that are produced byprotease activity and released into the media. Thus, activity ofgroup-transferring enzymes can be assayed readily using severalapproaches and many independent means of detection, which would beappreciated by one of ordinary skill in the art.

Protein chips can be used to identify proteins on the chip that havespecific activities such as specific kinases, proteases, nucleic acidbinding properties, nucleotide hydrolysis, hormone binding and DNAbinding. Thus, the chip can be probed with a probe that will indicatethe presence of the desired activity. For example, if DNA binding is theactivity of interest, the chip containing candidate DNA-binding proteinsis probed with DNA.

The search for probes (natural or synthetic) that are protein or nucleicacid ligands for an array of proteins can be carried out in parallel ona protein chip. A probe can be a cell, protein-containing cellularmaterial, protein, oligonucleotide, polynucleotide, DNA, RNA, smallmolecule substrate, drug candidate, receptor, antigen, steroid,phospholipid, antibody, immunoglobulin domain, glutathione, maltose,nickel, dihydrotrypsin, or biotin. Alternatively, the probe can be anenzyme substrate or inhibitor. For example, the probe can be a substrateor inhibitor of an enzyme chosen from the group consisting of kinases,phosphatases, proteases, glycosidases, acetylases, and other grouptransferring enzymes. After incubation of proteins on a chip withcombinations of nucleic acid or protein probes, the bound nucleic acidor protein probes can be identified by mass spectrometry (Lakey et al.,1998, “Measuring protein-protein interactions”, Curr Opin Struct Biol.8:119-23).

The identity of target proteins from pathogens (e.g., an infectiousdisease agent such as a virus, bacterium, fungus, or parasite) or targetproteins from abnormal cells (e.g., neoplastic cells, diseased cells, ordamaged cells) that serve as antigens in the immune response ofrecovering or non-recovering patients can be determined by using aprotein chip of the invention. For example, lymphocytes isolated from apatient can be used to screen protein chips comprising arrays of apathogen's proteins on a protein chip. In general, these screenscomprise contacting a positionally addressable array with a plurality oflymphocytes, said array comprising a plurality of potential antigens ona solid support, with each different antigen being at a differentposition on the solid support, wherein the density of different antigensis at least 100 different antigens per cm², and detecting positions onthe solid support where lymphocyte activation occurs. In a specificembodiment, lymphocytes are contacted with a pathogen's proteins on anarray, after which activation of B-cells or T-cells by an antigen or amixture of antigens is assayed, thereby identifying target antigensderived from a pathogen.

Alternatively, the protein chips are used to characterize an immuneresponse by, for example, screening arrays of potential antigens toidentify the targets of a patient's B-cells and/or T-cells. For example,B-cells can be incubated with an array of potential antigens (i.e.,molecules having antigenic determinants) to identify antigenic targetsfor humoral-based immunity. The source of antigens can be, for example,from autologous tissues, collections of known or unknown antigens (e.g.,of pathogenic microorganisms), tissue-specific or disease-specificantigen collections, or synthetic antigens.

In another embodiment, lymphocytes isolated from a patient can be usedto screen protein chips comprising arrays of proteins derived from apatient's own tissues. Such screens can identify substrates ofautoimmunity or allergy-causing proteins, and thereby diagnoseautoimmunity or allergic reactions, and/or identify potential targetdrug candidates.

In another embodiment, the protein chips of the invention are used toidentify substances that are able to activate B-cells or T-cells. Forexample, lymphocytes are contacted with arrays of test molecules orproteins on a chip, and lymphocyte activation is assayed, therebyidentifying substances that have a general ability to activate B-cellsor T-cells or subpopulations of lymphocytes (e.g., cytotoxic T-cells).

Induction of B-cell activation by antigen recognition can be assayed byvarious means including, but not limited to, detecting or measuringantibody synthesis, incorporation of ³H-thymidine, binding of labeledantibodies to newly expressed or suppressed cell surface molecules, andsecretion of factors indicative of B-cell activation (e.g., cytokines).Similarly, T-cell activation in a screen using a protein chip of theinvention can be determined by various assays. For example, a chromium(⁵¹Cr) release assay can detect recognition of antigen and subsequentactivation of cytotoxic T-cells (see, e.g., Palladino et al., 1987,Cancer Res. 47:5074-9; Blachere et al., 1993, J. Immunotherapy14:352-6).

The specificity of an antibody preparation can be determined through theuse of a protein chip of the invention, comprising contacting apositionally addressable array with an antibody preparation, said arraycomprising a plurality of potential antigens on a solid support, witheach different antigen being at a different position on the solidsupport, wherein the density of different antigens is at least 100different antigens per cm², and detecting positions on the solid supportwhere binding by an antibody in the antibody preparation occurs. Theantibody preparation can be, but is not limited to, Fab fragments,antiserum, and polyclonal, monoclonal, chimeric, single chain,humanized, or synthetic antibodies. For example, an antiserum can becharacterized by screening disease-specific, tissue-specific, or otheridentified collections of antigens, and determining which antigens arerecognized. In a specific embodiment, protein chip arrays having similaror related antigens are screened with monoclonal antibodies to evaluatethe degree of specificity by determining to which antigens on the arraya monoclonal antibody binds.

The identity of targets of specific cellular activities can be assayedby treating a protein chip with complex protein mixtures, such as cellextracts, and determining protein activity. For example, a protein chipcontaining an array of different kinases can be contacted with a cellextract from cells treated with a compound (e.g., a drug), and assayedfor kinase activity. In another example, a protein chip containing anarray of different kinases can be contacted with a cell extract fromcells at a particular stage of cell differentiation (e.g., pluripotent)or from cells in a particular metabolic state (e.g., mitotic), andassayed for kinase activity. The results obtained from such assays,comparing for example, cells in the presence or absence of a drug, orcells at several differentiation stages, or cells in different metabolicstates, can provide information regarding the physiologic changes in thecells between the different conditions.

Alternatively, the identity of targets of specific cellular activitiescan be assayed by treating a protein chip of the invention, containingmany different proteins (e.g., a peptide library), with a complexprotein mixture (e.g., such as a cell extract), and assaying formodifications to the proteins on the chip. For example, a protein chipcontaining an array of different proteins can be contacted with a cellextract from cells treated with a compound (e.g., a drug), and assayedfor kinase, protease, glycosidase, actetylase, phosphatase, or othertransferase activity, for example. In another example, a protein chipcontaining an array of different proteins can be contacted with a cellextract from cells at a particular stage of cell differentiation (e.g.,pluripotent) or from cells in a particular metabolic state (e.g.,mitotic). The results obtained from such assays, comparing for example,cells in the presence or absence of a drug, or cells at several stagesof differentiation, or cells in different metabolic states, can provideinformation regarding the physiologic effect on the cells under theseconditions.

The protein chips are useful to identify probes that bind to specificmolecules of biologic interest including, but not limited to, receptorsfor potential ligand molecules, virus receptors, and ligands for orphanreceptors.

The protein chips are also useful to detecting DNA binding or RNAbinding to proteins on the protein chips, and to determine the bindingspecificity. In addition, particular classes of RNA-binding orDNA-binding proteins (e.g., zinc-finger proteins) can be studied withthe protein chips by screening arrays of these proteins with nucleicacid sequences, and determining binding specificity and bindingstrength.

The identity of proteins exhibiting differences in function, ligandbinding, or enzymatic activity of similar biological entities can beanalyzed with the protein chips of the present invention. For example,differences in protein isoforms derived from different alleles areassayed for their activities relative to one another.

The high density protein chips can be used for drug discovery, analysisof the mode of action of a drug, drug specificity, and prediction ofdrug toxicity. For example, the identity of proteins that bind to adrug, and their relative affinities, can be assayed by incubating theproteins on the chip with a drag or drug candidate under different assayconditions, determining drug specificity by determining where on thearray the drug bound, and measuring the amount of drug bound by eachdifferent protein. Bioassays in which a biological activity is assayed,rather than binding assays, can alternatively be carried out on the samechip, or on an identical second chip. Thus, these types of assays usingthe protein chips of the invention are useful for studying drugspecificity, predicting potential side effects of drugs, and classifyingdrugs. Further, protein chips of the invention are suitable forscreening complex libraries of drug candidates. Specifically, theproteins on the chip can be incubated with the library of drugcandidates, and then the bound components can be identified, e.g., bymass spectrometry, which allows for the simultaneous identification ofall library components that bind preferentially to specific subsets ofproteins, or bind to several, or all, of the proteins on the chip.Further, the relative affinity of the drug candidates for the differentproteins in the array can be determined.

Moreover, the protein chips of the present invention can be probed inthe presence of potential inhibitors, catalysts, modulators, orenhancers of a previously observed interaction, enzymatic activity, orbiological response. In this manner, for example, blocking of thebinding of a drug, or disruption of virus or physiological effectors tospecific categories of proteins, can be analyzed by using a protein chipof the present invention.

The protein chips of the invention can be used to determine the effectsof a drug on the modification of multiple targets by complex proteinmixtures, such as for example, whole cells, cell extracts, or tissuehomogenates. The net effect of a drug can be analyzed by screening oneor more protein chips with drug-treated cells, tissues, or extracts,which then can provide a “signature” for the drug-treated state, andwhen compared with the “signature” of the untreated state, can be ofpredictive value with respect to, for example, potency, toxicity, andside effects. Furthermore, time-dependent effects of a drug can beassayed by, for example, adding the drug to the cell, cell extract,tissue homogenate, or whole organism, and applying the drug-treatedcells or extracts to a protein chip at various timepoints of thetreatment.

Screening of phage display libraries can be performed by incubating alibrary with the protein chips of the present invention. Binding ofpositive clones can be determined by various methods known in the art(e.g., mass spectrometry), thereby identifying clones of interest, afterwhich the DNA encoding the clones of interest can be identified bystandard methods (see, e.g., Ames et al., 1995, J. Immunol. Methods184:177-86; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-8;Persic et al., 1997, Gene 187:9-18). In this manner, the chips areuseful to select for cells having surface components that bind tospecific proteins on the chip. Alternatively, a phage display librarycan be attached to the chip, such that a positionally addressable arrayof the library is created, after which the array can be screenedrepeatedly with different mixtures of probes.

The invention also provides kits for carrying out the assay regimens ofthe invention. In a specific embodiment, kits of the invention compriseone or more arrays of the invention. Such kits may further comprise, inone or more containers, reagents useful for assaying biological activityof a protein or molecule, reagents useful for assaying interaction of aprobe and a protein or molecule, reagents useful for assaying thebiological activity of a protein or molecule having a biologicalactivity of interest, and/or one or more probes, proteins or othermolecules. The reagents useful for assaying biological activity of aprotein or molecule, or assaying interactions between a probe and aprotein or molecule, can be contained in each well or selected wells onthe protein chip. Such reagents can be in solution or in solid form. Thereagents may include either or both the proteins or molecules and theprobes required to perform the assay of interest.

In one embodiment, a kit comprises one or more protein chips (i.e.,positionally addressable arrays comprising a plurality of differentsubstances, selected from the group consisting of proteins, moleculescomprising functional domains of said proteins, whole cells, andprotein-containing cellular material, on a solid support, with eachdifferent substance being at a different position on the solid support),wherein the plurality of different substances consists of at least 100different substances per cm², and in one or more containers, one or moreprobes, reagents, or other molecules. The substances of the array can beattached to the surface of wells on the solid support. In anotherembodiment, the protein chip in the kit can have the protein or probealready attached to the wells of the solid support. In yet anotherembodiment, the protein chip in the kit can have the reagent(s) orreaction mixture useful for assaying biological activity of a protein ormolecule, or assaying interaction of a probe and a protein or molecule,already attached to the wells of the solid support. In yet anotherembodiment, the reagent(s) is not attached to the wells of the solidsupport, but is contained in the wells. In yet another embodiment, thereagent(s) is not attached to the wells of the solid support, but iscontained in one or more containers, and can be added to the wells ofthe solid support. In yet another embodiment, the kit further comprisesone or more containers holding a solution reaction mixture for assayingbiological activity of a protein or molecule. In yet another embodiment,the kit provides a substrate (e.g., beads) to which probes, proteins ormolecules of interest, and/or other reagents useful for carrying out oneor more assays, can be attached, after which the substrate with attachedprobes, proteins, or other reagents can be placed into the wells of thechip.

In another embodiment, one or more protein chips in the kit have,attached to the wells of the solid support, proteins with a biologicalactivity of interest. In another embodiment, one or more protein chipsin the kit have, attached to the wells of the solid support, at least50%, 75%, 90% or 95% of all expressed proteins with the same type ofbiological activity in the genome of an organism. In a specificembodiment, one or more protein chips in the kit have, attached to thewells of the solid support, at least 50%, 75%, 90% or 95% of allexpressed kinases, phosphatases, glycosidase, proteases, acetylases,other group transferring enzymes, nucleic acid binding proteins,hormone-binding proteins or DNA-binding proteins, within the genome ofan organism (e.g., of a particular species).

E. Proteins Useful with the Protein Chips

Full-length proteins, portions of full-length proteins, and peptideswhether prepared from recombinant overexpression in an organism,produced via fragmentation of larger proteins, or chemicallysynthesized, are utilized in this invention to form the protein chip.Organisms whose proteins are overexpressed include, but are not limitedto, bacteria, yeast, insects, humans, and non-human mammals such asmice, rats, cats, dogs, pigs, cows and horses. Further, fusion proteinsin which a defined domain is attached to one of a variety of natural orsynthetic proteins can be utilized. Proteins used in this invention canbe purified prior to being attached to, or deposited into, the wells ofthe protein chip, or purified during attachment via the use of reagentswhich have been previously attached to, or deposited into, the wells ofthe protein chip. These reagents include those that specifically bindproteins in general, or bind to a particular group of proteins. Proteinscan be embedded in artificial or natural membranes (e.g., liposomes,membrane vesicles) prior to, or at the time of attachment to the proteinchip. Alternatively, the proteins can be delivered into the wells of theprotein chip.

Proteins used in the protein chips of the present invention arepreferably expressed by methods known in the art. The InsectSelectsystem from Invitrogen (Carlsbad, Calif., catalog no. K800-01), anon-lytic, single-vector insect expression system that simplifiesexpression of high-quality proteins and eliminates the need to generateand amplify virus stocks, is a preferred expression system. Thepreferred vector in this system is pIB/V5-His TOPO TA vector (catalogno. K890-20). Polymerase chain reaction (PCR) products can be cloneddirectly into this vector, using the protocols described by themanufacturer, and the proteins are then expressed with N-terminalhistidine (His) labels which can be used to purify the expressedprotein.

The BAC-TO-BAC™ system, another eukaryotic expression system in insectcells, available from Lifetech (Rockville, Md.), is also a preferredexpression system. Rather than using homologous recombination, theBAC-TO-BAC™ system generates recombinant baculovirus by relying onsite-specific transposition in E. coli. Gene expression is driven by thehighly active polyhedrin promoter, and therefore can represent up to 25%of the cellular protein in infected insect cells.

Example 1 Analysis of Yeast Protein Kinases Using Protein Chips A.Introduction

The following example exemplifies the various aspects of protein chipproduction and a method of using the protein chips of the presentinvention. The protein chip technology of the present invention issuitable for rapidly analyzing large numbers of samples, and thereforethis approach was applied to the analysis of nearly all yeast proteinkinases. Protein kinases catalyze protein phosphorylation and play apivotal role in regulating basic cellular functions, such as cell cyclecontrol, signal transduction, DNA replication, gene transcription,protein translation, and energy metabolism⁷. The availability of acomplete genome sequence makes it possible to analyze all of the proteinkinases encoded by an organism and determine their in vitro substrates.

The yeast genome has been sequenced and contains approximately 6200 openreading frames greater than 100 codons in length; 122 of these arepredicted to encode protein kinases. Twenty-four of these protein kinasegenes have not been studied previously⁸. Except for two histidineprotein kinases, all of the yeast protein kinases are members of theSer/Thr family; tyrosine kinase family members do not exist althoughseven protein kinases that phosphorylate serine/threonine and tyrosinehave been reported⁸.

With the development of the protein chip technology of the presentinvention, the high throughput analysis of the biochemical activities ofnearly all of the protein kinases from Saccharomyces cerevisiae has beenconducted as described herein. Protein chips utilized were disposablearrays of 300 nl wells in silicone elastomer sheets placed on top ofmicroscope slides. The high density and small size of the wells allowsfor high throughput batch processing and simultaneous analysis of manyindividual samples, requiring only small amounts of protein. Usingprotein chips of the present invention, Saccharomyces cerevisiae kinaseproteins (119 different kinases in total) were fused toglutathione-Stransferase (GST), overexpressed in yeast, then purifiedand assayed for their ability to phosphorylate 17 different substrates.Nearly all of the kinases tested (93%) exhibited activities that were atleast five-fold higher than controls, on one or more substrates,including 18 of 24 previously uncharacterized kinases. Thirty-twokinases exhibited preferential phosphorylation of one or two substrates.Twenty-seven kinases readily phosphorylated poly(Tyr-Glu). Since onlyfive of these kinases were previously classified as dual functionkinases (i.e., they phosphorylate both Ser/Thr and Tyr), these findingsgreatly expand our knowledge as to which kinases are able tophosphorylate tyrosine residues. Interestingly, these dual specificitykinases often share common amino acid residues that lie near thecatalytic region. These results indicate that the protein chiptechnology of the present invention is useful for high throughputscreening of protein biochemical activity, and for the analysis ofentire proteomes.

B. Methods

1. Cell Culture, Constructs and Protein Purification

Using the recombination strategy of Hudson et al.⁹, 119 of 122 yeastprotein kinase genes were cloned into a high copy URA3 expression vector(pEG(KG)), which produces GST fusion proteins under the control of thegalactose-inducible GAL10 promoter¹⁰. Briefly, primers complementary tothe end of each ORF were purchased from Research Genetics; the ends ofthese primers contain a common 20 bp sequence. In a second round of PCR,the ends of these products were modified by adding sequences that arehomologous to the vector. The PCR products containing the vectorsequences at their ends were transformed along with the vector into apep4 yeast strain (which lacks several yeast proteases)¹⁰, and Ura⁺colonies were selected. Plasmids were rescued in E. coli, verified byrestriction enzyme digestion and the DNA sequence spanning thevector-insert junction was determined using a primer complementary tothe vector. For the GST::Cla4 construct, a frame-shift mutation wasfound in a poly(A) stretch in the amino terminal coding region. Threeindependent clones were required to find the correct one that maintainedreading frame. For five of these genes, two overlapping PCR productswere obtained and introduced into yeast cells. Confirmed plasmids werereintroduced into the pep4 yeast strain for kinase protein purification.

For preparing samples using the 96 well format, 0.75 ml of cells weregrown in medium containing raffinose to O.D.(600) about 0.5 in boxescontaining 2 ml wells; two wells were used for each strain. Galactosewas added to a final concentration of 4% to induce protein expression,and the cells were incubated for 4 hrs. The cultures of the same strainwere combined, washed once with 500 μl of lysis buffer, resuspended in200 μl of lysis buffer, and transferred into a 96×0.5 ml plate (DotScientific, USA) containing 100 μl chilled glass beads. Cells were lysedin the box by repeated vortexing at 4° C. and the GST fusion proteinswere purified from these strains using glutathione beads and standardprotocols²⁰ in a 96 well format. The purity of five purified GST::kinaseproteins (Swe1, Ptk2, Pkh1, Hog1, Pbs2) was determined by comparing theCoomasie staining patterns of the purified proteins with the patternsobtained by immunoblot analysis using anti-GST antibodies. The resultsindicated that the purified proteins were more than 90% pure. To purifythe activated form of Hog1, the cells were challenged with 0.4 M NaCl inthe last five minutes of the induction. Protein kinase activity wasstable for at least two months at −70° C. with little or no loss ofkinase activity.

2. Chip Fabrication and Protein Attachment

Chips were made from the silicone elastomer, polydimethylsiloxane (PDMS)(Dow Chemical, USA), which was cast over microfabricated molds. LiquidPDMS was poured over the molds and, after curing (at least 4 hours at65° C.), flexible silicone elastomer array sheets were then peeled fromthe reusable molds. Although PDMS can be readily cast overmicrolithographically fabricated structures, for the purposes of thekinase assay described herein, molds made from sheets of acrylicpatterned with a computer-controlled laser milling tool (Universal LaserSystems, USA) sufficed.

Over 30 different arrays were tested. The variables tested were widthand depth of the wells (widths ranging from 100 μm to 2.5 mm, depthsfrom 100 μm to 1 mm), spacing between wells (100 μm to 1 mm),configuration (either rectangular arrays or closest packed), and wellshape (square versus round). The use of laser milled acrylic moldsoffered a fast and inexpensive method to realize a large numberprototype molds of varying parameters.

To determine the conditions that maximize protein attachment to thewells, PDMS was treated with either 5 M H₂SO₄, 10 M NaOH, hydrogenperoxide or a 3-glycidooxypropyltrimethoxysilane linker (GPTS) (Aldrich,USA)^(11,12). GPTS treatment resulted in the greatest adsorption ofprotein to the wells relative to untreated. PDMS or PDMS treated otherways. Briefly, after washing with 100% EtOH three times at roomtemperature, the chips were immersed in 1% GPTS solution (95% EtOH, 16mM HOAc) with shaking for 1 hr at room temperature. After three washeswith 95% EtOH, the chips were cured at 135° C. for 2 hrs under vacuum.Cured chips can be stored in dry Argon for months¹². To attach proteinsto the chips, protein solutions were added to the wells and incubated onice for 1 to 2 hours. After rinsing with cold HEPES buffer (10 mM HEPES,100 mM NaCl, pH 7.0) three times, the wells were blocked with 1% BSA inPBS (Sigma, USA) on ice for >1 hr. Because of the use of GPTS, anyreagent containing primary amine groups was avoided.

To determine the concentration of proteins that can be linked to thetreated PDMS, horseradish peroxidase (HRP) anti-mouse Ig (Amersham, USA)was attached to the chip using serial dilutions of the enzyme. Afterextensive washing with PBS, the bound antibodies were detected using anenhanced chemiluminescent (ECL) detection method (Amersham, USA). Up to8×10⁻⁹ μg/μm² of protein can be attached to the surface; a minimum8×10⁻¹³ μg/μm² is required for detection by our immunostaining methods.

3. Immunoblotting, Kinase Assay and Data Acquisition

Immunoblot analysis was performed as described³⁴, GST::protein kinaseswere tested for in vitro kinase activity¹³ using ³³P-γ-ATP. In theautophosphorylation assay, the GST::kinases were directly adhered toGPTS-treated PDMS and the in vitro reactions carried out with ³³P-γ-ATPin appropriate buffer. In the substrate reactions, the substrate wasadhered to the wells via GPTS, and the wells were washed with HEPESbuffer and blocked with 1% BSA, before kinase, ³³P-γ-ATP and buffer wereadded. The total reaction volume was kept below 0.5 μl per reaction.After incubation for 30 minutes at 30° C., the chips were washedextensively, and exposed to both X-ray film and a Molecular DynamicsPhosphorImager, which has a resolution of 50 μm and is quantitative. Fortwelve substrates each kinase assay was repeated at least twice; for theremaining five, the assays were performed once.

To determine substrate specificity, specificity index (SI) wascalculated using the following formula: S_(ir)=F_(ir)/[(F_(i1)+F_(i2)+ .. . +F_(ir))/r], where i represents the ID of kinase used, r representsthe ID of a substrate, and F_(ir) represents the fold increase of akinase i on substrate r compared with GST alone.

4. Kinase Sequence Alignments and Phylogenetic Trees

Multiple sequence alignments based on the core kinase catalytic domainsubsequences of the 107 protein kinases were generated with the CLUSTALW algorithm³⁵, using the Gonnet 250 scoring matrix³⁶. Kinase catalyticdomain sequences were obtained from the SWISS-PROT³⁷, PIR³⁸, andGenBank³⁹ databases. For those kinases whose catalytic domains are notyet annotated (DBF4/YDR052C and SLN1/YIL147C), probable kinasesubsequences were inferred from alignments with other kinasesubsequences in the data set with the FASTA algorithm^(40,41) using theBLOSUM 50 scoring matrix⁴². Protein subsequences corresponding to theeleven core catalytic subdomains⁴³ were extracted from the alignments,and the phylogenetic trees were computed with the PROTPARS⁴⁴ program(FIG. 5 a).

5. Functional Grouping of Protein Chip Data

To visualize the approximate functional relationships between proteinkinases relative to the experimental data, kinases were hierarchicallyordered based on their ability to phosphorylate the 12 differentsubstrates (data available on the Internet atbioinfo.mbb.yale.edu/genome/yeast/chip as of Aug. 17, 2000). A profilecorresponding to the −/+ activity of the 107 protein kinases to each ofthe substrates was recorded, with discretized values in [0,1]. Matriceswere derived from the pairwise Hamming distances between experimentalprofiles, and unmated phylogenies were computed using theFitch-Margoliash least-squares estimation method⁴⁵ as implemented in theFITCH program34 of the PHYLIP software package⁴⁴. In each case, theinput order of taxa was randomized to negate any inherent bias in theorganization of the data set, and optimal hierarchies were obtainedthrough global rearrangements of the tree structures.

C. Results

1. Yeast Kinase Cloning and Protein Purification

Using a recombination-directed cloning strategy⁹, we attempted to clonethe entire coding regions of 122 yeast protein kinase genes in a highcopy expression vector (pEG(KG)) that produces GST fusion proteins underthe control of the galactose-inducible GAL10 promoters (FIG. 1 a).GST::kinase constructs were rescued into E. coli, and sequences at the5′-end of each construct were determined. Using this strategy, 119 ofthe 122 yeast protein kinase genes were cloned in-frame. The threekinase genes that were not cloned are very large (4.5-8.3 kb).

The GST:kinase fusion proteins were overproduced in yeast and purifiedfrom 50 ml cultures using glutathione beads and standard protocols¹¹.For the case of Hog1 the yeast cells were treated with high salt toactivate the enzyme in the last five minutes of induction; for the restof the kinases, synthetic media (URA⁻/raffinose) was used. Immunoblotanalysis of all 119 fusions using anti-GST antibodies revealed that 105of the yeast strains produced detectable GST::fusion proteins; in mostcases the fusions were full length. Up to 1 μg of fusion protein per mlof starting culture was obtained (FIG. 1 b). However, 14 of 119GST::kinase samples were not detected by immunoblotting analysis.Presumably, these proteins are not stably overproduced in the pep4protease-deficient strain used, or these proteins may form insolubleaggregates that do not purify using our procedures. Although thisprocedure was successful, purification of GST fusion proteins using 50ml cultures is a time-consuming process and not applicable for preparingthousands of samples. Therefore, a procedure for growing cells in a 96well format was developed (see Methods). Using this procedure, 119 GSTfusions were prepared and purified in six hours with about two-foldhigher yields per ml of starting culture relative to the 50 ml method.

2. Protein Chip Design

Protein chips were developed to conduct high throughput biochemicalassays of 119 yeast protein kinases (FIG. 2). These chips consist of anarray of wells in a disposable silicone elastomer polydimethylsiloxane(PDMS)¹¹. Arrays of wells allow small volumes of different probes to bedensely packed on a single chip yet remain physically segregated duringsubsequent batch processing. Proteins were covalently attached to thewells using a linker 3-glycidooxypropyltrimethoxysilane (GPTS)¹². Up to8×10⁻⁹ μg/μm² of protein can be attached to the surface (see Methods).

For the purposes of the protein kinase assays, the protein chiptechnology was configured to be compatible with standard sample handlingand recording equipment. Using radioisotope labeling (³³P), the kinaseassays described below, and manual loading, a variety of arrayconfigurations were tested. The following chips produced the bestresults: round wells with 1.4 mm diameter and 300 μm deep (approximately300 nl), in a 10×14 rectangular array configuration with a 1.8 mm pitch.A master mold of twelve of these arrays were produced, and a largenumber of arrays were repeatedly cast for the protein kinase analysis.Chips were placed atop microscope slides for handling purposes (FIG. 2a); the arrays covered slightly more than one third of a standardmicroscope slide and two arrays per slide were typically used (FIG. 2b). Although a manual pipette method to place proteins in each well wasemployed, automated techniques may also be used. In addition, thisprotein chip configuration may also be used with other labeling methods,such as by using fluorescently labeled antibodies to phosphoproteins,and subsequent detection of immunofluorescence.

3. Large-Scale Kinase Assays Using Protein Chips

All 119 GST::protein kinases were tested for in vitro kinase activity¹³in 17 different assays using ³³P-γ-ATP and 17 different chips. Each chipwas assayed using a different substrate, as follows: 1)Autophosphorylation, 2) Bovine Histone HI (a common kinase substrate),3) Bovine Casein (a common substrate), 4) Myelin basic protein (a commonsubstrate), 5) Ax12 C terminus-GST (Ax12 is a transmembranephosphoprotein involved in budding)¹⁴, 6) Rad9 (a phosphoproteininvolved in the DNA damage checkpoint)¹⁵, 7) Gic2 (a phosphoproteininvolved in budding)¹⁶, 8) Red1 (a meiotic phosphoprotein important forchromosome synapsis)¹⁷, 9) Mek1 (a meiotic protein kinase important forchromosome synapsis)¹⁸, 10) Poly(tyrosine-glutamate 1:4)(poly(Tyr-Glu)); a tyrosine kinase substrate)¹⁹, 11) Ptk2 (a smallmolecule transport protein)²⁰, 12) Hs11 (a protein kinase involved incell cycle regulation)²¹, 13) Swi6 (a phosphotranscription factorinvolved in G1/S control)²², 14) Tub4 (a protein involved in microtubulenucleation)²³, 15) Hog1 (a protein kinase involved in osmoregulation)²⁴,16) Hog1 (an inactive form of the kinase), and 17) GST (a control). Forthe autophosphorylation assay, the kinases were directly adhered to thetreated PDMS wells and ³³P-γ-ATP was added; for substrate reactions, thesubstrates were bound to the wells, and then kinases and ³³P-γ-ATP wereadded. After the reactions were completed, the slides were washed andthe phosphorylation signals were acquired and quantified using a highresolution phosphoimager. Examples are shown in FIG. 3. To identifykinase activities, the quantified signals were converted into foldincreases relative to GST controls and plotted for further analysis(FIG. 4 a-d).

As shown in FIG. 4 a-d, most (93.3%) kinases exhibited activityfive-fold or greater over background for at least one substrate. Asexpected, Hrr25, Pbs2 and Mek1 phosphorylated their knownsubstrates²⁵⁻²⁷, Swi6 (400-fold higher than the GST control), Hog1(10-fold higher) and Red1 (10-fold higher), respectively. The results ofthis assay demonstrated that 18 of the 24 predicted protein kinases havenot been studied previously phosphorylate one or more substrates, as doseveral unconventional kinases⁸, including the histidine kinases (Sln1,Yil042c) and phospholipid kinases (e.g., Mec1).

To determine substrate specificity, the activity of a particular kinasewas further normalized against the average of its activity against allsubstrates. Several examples are shown in FIG. 4 e-h; all the data areavailable on the Internet at bioinfo.mbb.yale.edu/genome/yeast/chip.Thirty-two kinases exhibited substrate specificity on a particularsubstrate with specificity index (SI; see Methods) equal or higher than2, and reciprocally, most substrates are preferentially phosphorylatedby a particular protein kinase or set of kinases. For example, the Cterminus of Ax12, protein involved in yeast cell budding, ispreferentially phosphorylated by Dbf20, Kin2, Yak1 and Ste20 relative toother protein. Interestingly, previous studies found that Ste20 waslocalized at the tip of emerging buds similar to Ax12, and aste20Δ/cla4^(ts) mutant is unable to bud or form fully polarized actinpatches or cables²⁸. Another example is the phosphoprotein Gic2, whichis also involved in budding¹⁶, Ste20 and Skm1 strongly phosphorylateGic2 (FIG. 4 e-h). Previous studies suggested that Cdc42 interacts withGic2, Cla4²⁹, Ste20 and Skm1. Our results raise the possibility thatCdc42 may function to promote the phosphorylation of Gic2 by recruitingSte20 and/or Skm1.

4. Yeast Contain Many Dual Specific Kinases

Of particular interest are the dual specificity kinases, i.e., thoseenzymes that phosphorylate both Ser/Thr and tyrosine. Based on sequenceanalysis, all but two yeast protein kinases belong to the Ser/Thr familyof protein kinases; however, at the time of the study, seven proteinkinases (Mps1, Rad53, Swe1, Ime2, Ste7, Hrr25, and Mck1) were reportedto be dual specificity kinases¹⁹. We confirmed that Swe1, Mps1, Ime2,and Hrr25 readily phosphorylate poly(Tyr-Glu), but we did not detect anytyrosine kinase activity for Ste7, Rad53 or Mck1. Mck1 did not showstrong activity in any of our assays; however, Ste7 and Rad53 are veryactive in other assays. Thus, their inability to phosphorylatepoly(Tyr-Glu) indicates that they are either very weak tyrosine kinasesin general or at least are weak with the poly(Tyr-Glu) substrate.Consistent with the latter possibility, others have found thatpoly(Tyr-Glu) is a very poor substrate for Rad53 (Ref 19; D. Stem, pers.comm.). Interestingly, we found that 23 other kinases also efficientlyuse poly(Tyr-Glu) as a substrate, indicating that there are at least 27kinases in yeast that are capable of acting in vitro as dual specificitykinases. One of these, Rim1, was recently shown to phosphorylate a Tyrresidue on its in vivo substrate, Ime2, indicating that it is a bonafide dual specificity kinase³⁰. In summary, this experiment roughlytripled the number of kinases capable of acting as dual specificitykinases, and has raised questions about some of those classified as suchkinases.

5. Correlation Between Functional Specificity and Amino Sequences of thePoly(Tyr-Glu) Kinases

The large-scale analysis of yeast protein kinases allows us to comparethe functional relationship of the protein kinases to one another. Wefound that many of the kinases that phosphorylate poly(Tyr-Glu) arerelated to one another in their amino acid sequences: 70% of thepoly(Tyr-Glu) kinases cluster into distinct four groups on a dendrogramin which the kinases are organized relative to one another based onsequence similarity of their conserved protein kinase domains (FIG. 5a). Further examination of the amino acid sequence reveals four types ofamino acids that are preferentially found in the poly(TyrGlu) class ofkinases relative to the kinases that do not use poly(Tyr-Glu) as, asubstrate (three are lysines and one is a methionine); one residue (anasparagine) was preferentially located in the kinases that do notreadily use poly(Tyr-Glu) as a substrate (FIG. 5 b). Most of theresidues lie near the catalytic portion of the molecule (FIG. 5 b)³¹,suggesting that they may play a role in substrate recognition.

D. Discussion

1. Large-Scale Analysis of Protein Kinases

This study employed a novel protein chip technology to characterize theactivities of 119 protein kinases for 17 different substrates. We foundthat particular proteins are preferred substrates for particular proteinkinases, and vice versa, many protein kinases prefer particularsubstrates. One concern with these studies is that it is possible thatkinases other than the desired enzyme are contaminating ourpreparations. Although this cannot be rigorously ruled out, analysis offive of our samples by Coomasie staining and immunoblot staining withanti-GST does not reveal any detectable bands in our preparation thatare not GST fusions (see methods).

It is important to note that in vitro assays do not ensure that asubstrate for a particular kinase in vitro is phosphorylated by the samekinase in vivo. Instead, these experiments indicate that certainproteins are capable of serving as substrates for specific kinases,thereby allowing further analysis. In this respect, these assays areanalogous to two-hybrid studies in which candidate interactions aredetected. Further experimentation is necessary to determine if theprocesses normally occur in vivo.

Consistent with the idea that many of the substrates are likely to bebona fide substrates in vivo is the observation that three kinases,Hrr25, Pbs2 and Mek1, phosphorylate their known substrates in ourassays. Furthermore, many of the kinases (e.g., Ste20) co-localize withtheir in vitro substrates (e.g., Ax12). Thus, we expect many of thekinases that phosphorylate substrates in our in vitro assays are likelyalso to do so in vivo.

Although most of the kinases were active in our assays, several werenot. Presumably, our preparations of these latter kinases either lacksufficient quantities of an activator or were not purified underactivating conditions. For example, Cdc28 which was not active in ourassays, might be lacking its activating cyclins. For the case of Hog1,cells were treated with high salt to activate the enzyme. Since nearlyall of our kinase preparations did exhibit activity, we presume that atleast some of the enzyme in the preparation has been properly activatedand/or contains the necessary cofactors. It is likely that theoverexpression of these enzymes in their native organism contributessignificantly to the high success of obtaining active enzymes.

Using the assays on the protein chip, many kinases that utilizepoly(Tyr-Glu) were identified. The large-scale analysis of many kinasesallowed the novel approach of correlating functional specificity ofpoly(Tyr-Glu) kinases with specific amino acid sequences. Many of theresidues of the kinases that phosphorylate poly(Tyr-Glu) contain basicresidues. This might be expected if there were electrostaticinteractions between the kinases residues and the Glu residues. However,the roles of some of the other residues are not obvious such as the Metresidues on the kinases that phosphorylate poly(Tyr-Glu) and the Asn onthose that do not. These kinase residues may confer substratespecificity by other mechanisms. Regardless, analysis of additionalsubstrates should allow further correlation of functional specificitywith protein kinase sequence for all protein kinases.

2. Protein Chip Technology

In addition to the rapid analysis of large number of samples, theprotein chip technology described here has significant advantages overconventional methods. 1) The chip-based assays have high signal-to-noiseratios. We found that the signal-to-noise ratio exhibited using theprotein chips is much better (>10 fold) than that observed fortraditional microtiter dish assays (data not shown). Presumably this isdue to the fact that ³³P-γ-ATP does not bind the PDMS as much asmicrotiter dishes. 2) The amount of material needed is very small.Reactions volumes are 1/20-1/40 the amount used in the 384-wellmicrotiter dishes; less than 20 ng of protein kinase was used in eachreaction. 3) The enzymatic assays using protein chips are extremelysensitive. Even though only 105 fusions were detectable by immunoblotanalysis, 112 exhibited enzymatic activity greater than five-fold overbackground for at least one substrate. For example, Mps1 consistentlyexhibits the strongest activity in many of the kinase assays even thoughwe have not been able to detect this fusion protein by immunoblotanalysis (see FIGS. 1 b and 3 a). 4) Finally, the chips are inexpensive;the material costs less than eight cents for each array. Themicrofabricated molds are also easy to make and inexpensive.

In addition to the analysis of protein kinases, this protein chiptechnology is also applicable to a wide variety of additional assays,such as ATP and GTP binding assays, nuclease assays, helicase assays andprotein-protein interaction assays. Recently, in an independent study,Phizicky and coworkers expressed yeast proteins as GST fusions under themuch weaker CUP1 promotor⁶. Although the quality of their clones has notbeen established, they were able to identify biochemical activitiesusing pools of yeast strains containing the fusion proteins. Theadvantage of our protein chip approach is that all samples can beanalyzed in a single experiment. Furthermore, although this study usedwells which have the advantage of segregating samples, flat PDMS chipsand glass slides can also be used for different assays; these have theadvantage that they can be used with standard pinning toolmicroarrayers. This technology can also be applied to facilitatehigh-throughput drug screening in which one can screen for compoundsthat inhibit or activate enzymatic activities of any gene products ofinterest. Since these assays will be carried out at the protein level,the results will be more direct and meaningful to the molecular functionof the protein.

We configured the protein chip technology for a specific protein kinaseassay using commonly available sample handling and recording equipment.For this purpose, array dimensions remained relatively large compared todimensions readily available with microfabricated silicone elastomerstructures³², We have cast PDMS structures with feature sizes two ordersof magnitude smaller than those reported here usingmicrolithographically fabricated molds, while others have reportedsubmicron feature sizes in microfabricated structures³³. These resultsindicate that well densities of microfabricated protein chips can bereadily increased by several orders of magnitude. The protein chiptechnology reported here is readily scalable.

In conclusion, an inexpensive, disposable protein chip technology wasdeveloped for high throughput screening of protein biochemical activity.Utility was demonstrated through the analysis of 119 protein kinasesfrom Saccharomyces cerevisiae assayed for phosphorylation of 17different substrates. These protein chips permit the simultaneousmeasurement of hundreds of protein samples. The use of microfabricatedarrays of wells as the basis of the chip technology allows arraydensities to be readily increased by several orders of magnitude. Withthe development of appropriate sample handling and measurementtechniques, these protein chips can be adapted for the simultaneousassay of several thousand to millions of samples.

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Example II Analysis of Yeast Protein Kinase Activity Using Protein ChipsA. Introduction

The following example presents three protocols that, for illustrationpurposes only, provide different methods of using the protein chips ofthe present invention to assay for protein kinase activity.

1. Assay Methods for Protein Kinase Activity

i. Autophosphorylation Activity

(1) Protein chips were washed three times with 100% EtOH at roomtemperature. The chips were then coated with the linker GPTS (1% in 95%EtOH) at room temperature for one hour with shaking. After washing with100% EtOH three times, the chips were dried at 130° C. for 1.5 hoursunder vacuum.

(2) GST::yeast protein kinases, one kinase species per well, were boundto the wells of the protein chip by incubation for at least one hour.The chip was further blocked by 1% BSA.

(3) Kinase buffer and a ³³P-γ-ATP probe was added to each well, andincubated at 30° C. for 30 minutes. The chip was washed extensivelyafter the phosphorylation reaction was completed.

(4) The specific ³³P-γ-ATP signal, representing autophosphorylation, wasdetected and quantified by a phosphoimager.

ii. Kinase Activity—Protocol I

(1) Protein chips were washed three times with 100% EtOH at roomtemperature. The chips were then coated with the linker GPTS (1% in 95%EtOH) at room temperature for one Lour with shaking. After washing with100% EtOH three times, the chips were dried at 130° C. for 1.5 hoursunder vacuum.

(2) A substrate (for example, GST::yeast protein) was bound to the chipsby incubation for one or more hours. The chip was further blocked by 1%BSA, and the chip was washed.

(3) A different protein kinase was added to each well of the proteinchip, along with kinase buffer and ³³P-γ-ATP, and incubated at 30° C.for 30 minutes. The protein chip was washed extensively after thephosphorylation reaction was completed.

(4) The specific ³³P-γ-ATP signal, representing phosphorylation of thesubstrate protein by the protein kinase probe, was detected andquantified by a phosphoimager.

iii. Kinase Activity—Protocol II

(1) Protein chips were washed three times with 100% EtOH at roomtemperature. The chips were then coated with the linker GPTS (1% in 95%EtOH) at room temperature, for one hour with shaking. After washing with100% EtOH three times, the chips were dried at 130° C. for 1.5 hoursunder vacuum.

(2) A substrate (for example, GST::yeast protein) was bound to the chipsby incubation for one or more hours. The chip was further blocked by 1%BSA and the chip was washed.

(3) A different protein kinase was added to each well of the proteinchip, along with kinase buffer and P-γ-ATP, and incubated at 30° C. for30 minutes. The protein chip was washed extensively after thephosphorylation reaction was completed. The chip was incubated withiodoacetyl-LC-biotin in the dark at room temperature overnight.

(4) After washing, the chip was probed with fluorescent-labeled avidinto detect the phosphorylation signals,

(5) The chip was then scanned using an Axon Genepix 4000A scanner, whichwas modified with a lens having an increased depth of focus of about300-400 microns. The modifications allow scanning of surfaces mounted ona slide (e.g., the PDMS microarrays of the present invention), whichwould otherwise be out of the plane of focus. Using the modified AxonGenepix 4000A scanner, the arrays were scanned to acquire and quantifyfluorescent signals.

Example III Analysis of Protein-Protein Interactions Using Protein Chips

A protein of interest (“probe protein”) is recombinantly expressed inand purified from E. coli as a labeled fusion protein using standardprotocols. The target proteins are attached to the wells of the chip,with a different target protein in each well. The purified probe proteinis introduced into each well of the chip, and incubated for severalhours or more. The chip is washed and probed with either: a) antibodiesto the probe protein, or b) antibodies to the label on the fusionprotein. The antibodies are labeled with a fluorescent label, such asCy3 or Cy5, or are detected using a fluorescently labeled secondaryantibody that detects the first antibody.

The following examples provide, for illustration purposes only, methodsof using the protein chips of the present invention to assay forproteases, nucleases, or G-protein receptors. Protein-proteininteractions generally can be assayed using the following or a similarmethod.

A. Analysis of Protease Activity

Protease activity is assayed in the following way. First, protein probesare prepared consisting of various combinations of amino acids, with aC-terminal or N-terminal mass spectroscopic label attached, with theonly proviso being that the molecular weight of the label should besufficiently large so that all labeled cleavage products of the proteincan be detected. The protein probe is contacted with proteases attachedto a protein chip at 37° C. After incubation at 37° C. for anappropriate period of time, and washing with acetonitrile andtrifluoroacetic acid, protease activity is measured by detecting theproteolytic products using mass spectrometry. This assay providesinformation regarding both the proteolytic activity and specificity ofthe proteases attached to the protein chip.

Another rapid assay for protease activity analysis is to attach proteinsof known sequence to the chip. The substrate proteins are fluorescentlylabeled at the end not attached to the chip. Upon incubation with theprotease(s) of interest, the fluorescent label is lost upon proteolysis,such that decreases in fluorescence indicate the presence and extent ofprotease activity. This same type of assay can be carried out whereinthe protein substrates are attached to beads placed in the wells of thechips.

B. Analysis of Nuclease Activity

Nuclease activity is assessed in the same manner as described forprotease activity, above, except that nucleic acid probes/substrates aresubstituted for protein probes/substrates. As such, fluorescently taggednucleic acid fragments that are released by nuclease activity can bedetected by fluorescence, or the nucleic acid fragments can be detecteddirectly by mass spectrometry.

C. Analysis of G-Protein Coupled Receptors

In another type of assay, compounds that bind G-protein coupledreceptors are identified. Initially, the G-protein receptor is cloned asa GST fusion protein, with the GST portion attached to the C terminus ofthe G-protein because the C-terminus is generally not involved withdetermining probe specificity. The G-protein::GST fusion proteins areattached to the wells, preferably by association with glutathione. TheG-protein receptors are then incubated with a mixture of compounds, suchas a combinatorial chemical library or a peptide library. After washing,bound probes are eluted, for example by the addition of 25%acetonitrile/0.05% trichloroacetic acid. The eluted material is then beloaded into a MALDI mass spectrometer and the nature of the bound probesidentified.

Example IV Analysis of Protein Kinases Inhibition by Specific InhibitorsUsing Protein Chips

The following description provides, for exemplary purposes only, methodsof using the protein chips of the present invention to examine proteinkinases for sensitivity to protein kinase inhibitors. Protein-proteininteractions generally can be assayed using the following or similarmethod.

Substrates were bound to the surface of the GPTS-treated microwells onthe protein chip at room temperature for one hour, then blocked with 1%BSA and 100 mM Tris pH 7.5, and washed three times with TBS buffer.Kinases and different concentrations of kinase inhibitors were added tothe microwells in the presence of ³³P-γ-ATP. The phosphorylationreaction was carried out at 30° C. for thirty minutes. After completionof the reaction, the protein chip was washed extensively with TBS bufferat room temperature, and then allowed to dry. Phosphorylation signalswere obtained by exposing the protein chip to either X-ray film or aphosphoimager.

A human protein kinase A (PKA), a human map kinase (MAPK), three yeastPKA homologs (TPK1, TPK2 and TPK3), and two other yeast protein kinases(HSL1 and RCK1) were tested against two substrates (i.e., a proteinsubstrate for PKA and a commonly used kinase substrate, MBP) usingdifferent concentrations of PKIα (a specific human PKA inhibitor) orSB202190 (a MAPK inhibitor). As shown in FIG. 7, PKIα specificallyinhibited PKA activities on both peptide and MBP substrates. However,PKIα did not inhibit the three yeast PKA homologs (TPK1, TPK2, TPK3) orthe other two yeast protein kinases tested, HSL1 and RCK1). In addition,SB202190 did not inhibit PKA activity.

Example V Kinase Assays on a Glass Surface

1. Glass slides (Fisher, USA) were soaked in 28-30% ammonium hydroxideovernight at room temperature (“RT”) with shaking.

2. The slides were rinsed with ultra-pure water four times for 5 minutes(“min”) each, then rinsed with a large volume of 100% ethanol (“EtOH”)to completely remove the water. Slides were then rinsed with 95% ethanolthree times.

3. The slides were immersed in 1% 3-glycidoxypropyltrimethoxysilane(GPST) solution in 95% EtOH, 16 mM acetic acid (“HOAc”) with shaking for1 hr at room temperature. The slides were rinsed with 95% ethanol threetimes at RT.

4. The slides were cured at 135° C. for 2 hrs under vacuum. Aftercooling, the slides can be stored in Argon for months before use.

5. Approximately 10 μl of each protein substrate (in 40% glycerol) werearrayed onto a 96-well PCR plate on ice. A manual spotting device (V&PScientific, USA) was used to spot approximately 3 nl of each of thesamples onto the GPTS-treated glass slide at RT. In one embodiment, 768samples are spotted on a single slide. The slides were incubated in acovered and clean chamber at RT for one hour.

6. A slide was blocked with 10 ml blocking buffer (100 mM glycine, 100mM Tris, pH 8.0, 50 mM NaCl) at RT for one hour. The slides were washedwith TBS buffer (50 mM Tris, pH 8.0, 150 mM NaCl) three times and spunto dryness at 1500 rpm for 5 min.

7. The substrate surfaces on the slides were covered with the HybriWellSealing System (Schleicher & Schuell, Germany) and 40 μl of kinasemixture, containing a protein kinase and ³³P-γ-ATP as a labelingreagent, was added to the substrates on ice.

8. The reaction was incubated at 30° C. for 30 min in a humiditychamber. The seals were peeled from the slides, and the slides immersedinto large volume of PBS buffer containing 50 mM EDTA. The slides werefurther washed with the same buffer 3×15 min at RT. The washed slideswere then dried with Kimwipes.

9. To acquire the signals, the slides were exposed to a Phosphoimagerscreen and the data analyzed using ImageQuant software.

REFERENCES CITED

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A positionally addressable array comprising aplurality of different substances on a solid support, with eachdifferent substance being at a different position on the solid support,wherein the density of the different substances on the solid support isat least 100 different substances per cm², and wherein the plurality ofdifferent substances comprises at least 61 purified active kinases orfunctional kinase domains thereof of a mammal, 61 purified activekinases or functional kinase domains thereof of a yeast, or 61 purifiedactive kinases or functional kinase domains thereof of a Drosophila. 2.The array of claim 1 wherein the density of the different substances onthe array is between 100 and 1,000 different substances per cm².
 3. Thearray of claim 1 wherein the density of the different substances on thearray is between 1,000 and 10,000 different substances per cm².
 4. Thearray of claim 1 wherein the density of the different substances on thearray is between 10,000 and 100,000 different substances per cm².
 5. Thearray of claim 1 wherein the density of the different substances on thearray is between 100,000 and 1,000,000 different substances per cm². 6.The array of claim 1 wherein the density of the different substances onthe array is between 1,000,000 and 10,000,000 different substances percm².
 7. The array of claim 1 wherein the density of the differentsubstances on the array is between 10,000,000 and 25,000,000 differentsubstances per cm².
 8. The array of claim 1 wherein the density of thedifferent substances on the array is at least 25,000,000 differentsubstances per cm².
 9. The array of claim 1 wherein the density of thedifferent substances on the array is at least 10,000,000,000 differentsubstances per cm².
 10. The array of claim 1 wherein the density of thedifferent substances on the array is at least 10,000,000,000,000different substances per cm².
 11. The array of claim 1 wherein theplurality of different substances are attached to the solid support viaa 3-glycidoxypropyl-trimethoxysilane linker.
 12. The array of claim 1wherein the mammal is selected from the group consisting of human,primate, mouse, rat, cat, dog, horse, and cow.
 13. The array of claim12, wherein the organism is mouse.
 14. The array of claim 12, whereinthe organism is mouse.
 15. The array of claim 12, wherein the organismis rat.
 16. The positionally addressable protein array of claim 1,wherein the plurality of different substances comprises 61 differentpurified active kinases of an organism.
 17. The positionally addressableprotein array of claim 1, wherein the plurality of different substancescomprises 92 different purified active kinases of a mammal, a yeast, ora Drosophila.
 18. The positionally addressable protein array of claim 1,wherein the plurality of different substances comprises 110 differentpurified active kinases of a mammal, a yeast, or a Drosophila.
 19. Thepositionally addressable protein array of claim 1, wherein the pluralityof different substances comprises 116 different purified active kinasesof a mammal, a yeast, or a Drosophila.
 20. The positionally addressableprotein array of claim 1, wherein the plurality of different substancescomprises 119 different purified active kinases of a mammal, a yeast, ora Drosophila.
 21. The positionally addressable protein array of claim 1,wherein the plurality of different substances comprises 122 purifiedactive different kinases of a mammal, a yeast, or a Drosophila.
 22. Thepositionally addressable array of claim 1, wherein the kinases are yeastkinases.
 23. The positionally addressable array of claim 1, wherein thedifferent substances are 61 purified active kinases.
 24. Thepositionally addressable array of claim 23, wherein the kinases areserine/threonine kinase family members, tyrosine kinase family members,or serine/threonine kinase and tyrosine kinase family members.
 25. Thepositionally addressable array of claim 1, wherein the functional kinasedomains are functional kinase domains of serine/threonine kinase familymembers, functional kinase domains of tyrosine kinase family members, orfunctional kinase domains of serine/threonine kinase family members orfunctional kinase domains of tyrosine kinase family members.
 26. Thepositionally addressable array of claim 23, wherein the 61 purifiedactive kinases are at least 100 amino acids in length.